Induction of SOCS-3 is insufficient to confer IRS-1 protein degradation in 3T3-L1 adipocytes

Insulin receptor substrate (IRS)-1 is a key protein in insulin signaling. Several studies have shown that the expression of IRS-1 can be modulated by protein degradation via the proteasome and the degradation of IRS-1 can be related to insulin-resistant states. The degradation of IRS-1 has been shown to be induced by SOCS-1 and SOCS-3 via the ubiquitin pathway. The goal of our study was to determine if the induction of SOCS-3 correlated with increased IRS-1 degradation in cultured 3T3-L1 adipocytes. Interestingly, our studies have shown that there is little correlation between the induction in SOCS-3 expression and the degradation of IRS-1 in mature 3T3-L1 adipocytes. Our results clearly demonstrate that treatment with leukemia inhibitory factor (LIF) or cardiotrophin (CT)-1 strongly induces the expression of SOCS-3 in mature 3T3-L1 adipocytes, but does not affect the degradation of IRS-1. On the contrary, tumor necrosis factor (TNF) alpha and insulin, which very weakly induce SOCS-3 expression, have profound effects on IRS-1 degradation. In summary, our results indicate that the expression of SOCS-3 does not correlate with the degradation of IRS-1 proteins in fat cells.     

Seeing the trees in the forest: selective electroporation of adipocytes within adipose tissue

Electroporation has been recently adapted for the transfer of macromolecules into cells of tissues in vivo. Although mature adipocytes constitute <20% of cells residing in adipose tissue, we hypothesized that fat cells might be susceptible to selective electrotransfer of plasmid DNA owing to their large size relative to other cells in the tissue. Results demonstrate the feasibility of electroporating DNA into mature fat cells with >99% selectivity over other cells in the tissue. Further experiments used the "adiporation" technique to image the subcellular targeting of fluorescent bioreporter molecules to the nucleus, mitochondria, and lipid droplets of adipocytes within intact adipose tissue. Finally, we utilized fluorescent bioreporters to examine the effects of constitutive activation of the beta-adrenergic signaling pathway in adipocytes. These results demonstrate that overexpression of rat beta1-adrenergic receptors alters the cellular morphology of white adipocytes in a fashion that mimics the effects of systemic infusion of beta3-adrenergic receptor agonists. Hallmarks of the altered morphology include pronounced fragmentation of the single lipid droplet, repositioning of the nucleus, and induction of mitochondrial biogenesis. These results indicate that activation of beta-adrenergic signaling within adipocytes is sufficient to induce a phenotype that resembles typical brown adipocytes and suggest that in vivo electroporation will allow molecular dissection of the mechanisms involved.     

Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.     

Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism

Obesity is an alarming primary health problem and is an independent risk factor for type II diabetes, cardiovascular diseases, and hypertension. Although the pathologic mechanisms linking obesity with these co-morbidities are most likely multifactorial, increasing evidence indicates that altered secretion of adipose-derived signaling molecules (adipokines; e.g. adiponectin, leptin, and tumor necrosis factor alpha) and local inflammatory responses are contributing factors. Chemerin (RARRES2 or TIG2) is a recently discovered chemoattractant protein that serves as a ligand for the G protein-coupled receptor CMKLR1 (ChemR23 or DEZ) and has a role in adaptive and innate immunity. Here we show an unexpected, high level expression of chemerin and its cognate receptor CMKLR1 in mouse and human adipocytes. Cultured 3T3-L1 adipocytes secrete chemerin protein, which triggers CMKLR1 signaling in adipocytes and other cell types and stimulates chemotaxis of CMKLR1-expressing cells. Adenoviral small hairpin RNA targeted knockdown of chemerin or CMKLR1 expression impairs differentiation of 3T3-L1 cells into adipocytes, reduces the expression of adipocyte genes involved in glucose and lipid homeostasis, and alters metabolic functions in mature adipocytes. We conclude that chemerin is a novel adipose-derived signaling molecule that regulates adipogenesis and adipocyte metabolism.     

Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential

When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.     

Programming human pluripotent stem cells into white and brown adipocytes

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.